Pseudomontella Z3-4 for inducing tobacco bacterial wilt immunity and application of pseudomontella Z3-4

Abstract

The invention discloses Pseudomonas mendogensis Z3-4 capable of inducing tobacco bacterial wilt immunity and application of the Pseudomonas mendogensis Z3-4. The Pseudomonas mendogensis Z3-4 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation address is Beijing, the preservation center number is CGMCC No.35660, the preservation date is August 18, 2025, and the Pseudomonas mendogensis Z3-4 is classified and named as Pseudomonas mendogensis. The strain Z3-4 is an efficient tobacco root colonization bacterium, and can significantly induce the immunocompetence of tobacco to bacterial wilt. The strain Z3-4 is used as a tobacco bacterial wilt resistant vaccine, is applied by adopting a root irrigation method, can effectively prevent and treat tobacco bacterial wilt, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a strain of Pseudomonas moniliforme Z3-4 that induces immunity to tobacco bacterial wilt and its application, belonging to the field of tobacco bacterial wilt disease control. Background Technology

[0002] Tobacco, as one of my country's important economic crops, occupies a pivotal position in the agricultural industrial structure. Statistics show that the planting area of ​​tobacco has consistently remained above 20% of the total area of ​​economic crops. However, continuous cropping of tobacco for many years often leads to the continuous deterioration of the soil environment, most notably manifested in the explosive growth of soil-borne diseases. Bacterial wilt, a representative disease, is the most severe, causing yield losses of 30%-50%, seriously restricting the improvement of tobacco yield and quality.

[0003] Currently, the commonly used soil improvement measures in tobacco-growing areas mainly include chemical fungicide treatment and soil fumigation. These traditional measures have significant limitations in practical application. While chemical fungicides or soil fumigation can effectively inhibit pathogens in the short term, long-term use leads to drug resistance in pathogens, eliminates a large number of beneficial microorganisms, disrupts the balance of the soil microbial community, and may cause pesticide residue problems, which are detrimental to agricultural production. Against this backdrop, biological control technology has shown great application potential, providing a new solution to the problem of soil-borne diseases in tobacco. From an ecological perspective, biocontrol microorganisms can inhibit pathogen reproduction through nutrient competition, spatial occupation, and the growth promotion of metabolic products, thereby rebuilding a healthy soil micro-ecosystem. However, general biocontrol agents only control the occurrence of soil-borne diseases from the perspective of biological competition, often with unstable effects. Therefore, the ideal biocontrol bacterial formulation should break through the traditional mindset of killing pathogens and instead focus on enhancing the plant's intrinsic immunity. Its core lies in activating the plant's own immune system by regulating the plant-microbe interaction to achieve disease control.

[0004] Currently, the application of biocontrol bacteria to enhance the plant immune system has made some progress. Certain biocontrol bacteria can optimize the intensity of the immune response without causing disease, forming a "vaccine"-like sustained protective effect and developing systemic resistance, providing a new solution for the sustainable development of the tobacco industry. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Pseudomonas moniliforme Z3-4 that induces immunity to tobacco bacterial wilt and its application, which can effectively inhibit the occurrence of tobacco bacterial wilt.

[0006] Technical solution: To solve the above technical problems, the present invention provides a strain of Pseudomonas mandelii Z3-4, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 35660 and deposit date of August 18, 2025. Its classification name is Pseudomonas mandelii.

[0007] The 16S rDNA base sequence of the strain is shown in SEQ ID No: 1.

[0008] The present invention also provides a bacterial agent or preparation containing one or more of the aforementioned Pseudomonas mandelii Z3-4 or its fermentation broth or bacterial suspension.

[0009] The content of Pseudomonas mandelii Z3-4 was 5 × 10⁻⁶. 7 -1×10 8 cfu / ml.

[0010] The present invention also provides the application of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned bacterial agent or preparation in the control of tobacco bacterial wilt disease.

[0011] Among these methods, the prevention and control of tobacco bacterial wilt is achieved by activating the tobacco's immunity against bacterial wilt.

[0012] The methods for preventing and controlling tobacco bacterial wilt include applying the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned bacterial agent or preparation to tobacco.

[0013] Methods for preventing and controlling tobacco bacterial wilt include soaking tobacco seeds or drenching the roots of tobacco plants.

[0014] The aforementioned tobacco bacterial wilt disease is caused by Ralstonia solanacearum or its variants.

[0015] The present invention also provides the use of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned bacterial agent or preparation in the preparation of products for the prevention and control of tobacco bacterial wilt.

[0016] The products mentioned include vaccines.

[0017] The present invention also provides the application of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned bacterial agent or preparation in inducing tobacco root defense metabolism.

[0018] The present invention also provides the application of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned bacterial agent or preparation in improving the immunity of tobacco against tobacco bacterial wilt disease.

[0019] Among them, Pseudomonas mandelii Z3-4 can activate tobacco's immunity against bacterial wilt and promote the application of tobacco seedling disease control.

[0020] The present invention also provides a method for obtaining the vaccine strain:

[0021] (1) Collect intact roots of disease-resistant tobacco in the field and obtain aseptic roots on the surface.

[0022] (2) The aseptic roots were ground into powder using a sterilized mortar and pestle, and then diluted with PBS to obtain a root dilution. The root dilution was then spread on TSA agar plates. After incubation, single colonies were picked from plates with appropriate concentration gradients according to bacterial morphology, color and size and streaked on the plates for purification. The strains obtained after purification were used as test strains and inoculated into TSA agar for temporary preservation.

[0023] (3) Select a single colony and streak it on a plate to purify it. Repeat the purification process three times.

[0024] (4) Extract DNA from the tested strains and perform 16S rRNA sequencing to determine the taxonomic position of the strains.

[0025] This invention also provides a method for determining the root colonization effect of a bacterial strain through a sterile seedling colonization test:

[0026] (1) Prepare MS medium with 1 / 2 strength: Dissolve Murashige Skoog medium (containing vitamins) dry powder with sucrose and agar powder in sterile deionized water, adjust the pH to 5.8, and autoclave.

[0027] (2) Tobacco seedling culture: Tobacco seeds were surface sterilized and then placed in MS medium with a strength of 1 / 2. After germination, the seedlings were transferred to MS medium for further culture.

[0028] (3) Preparation of Z3-4 fermentation broth: Strain Z3-4 was fermented for 2 days, and the bacterial concentration was adjusted to OD600 = 0.1 with sterile water. Each plant was inoculated with the bacterial suspension, and the bacteria were recovered weekly. The tobacco roots were rinsed with sterile water to remove floating bacteria. Then, the surface microorganisms of the tobacco roots were removed; the remaining roots were soaked in 75% alcohol, then in 3% NaClO solution, and finally rinsed with sterile water. After being ground in a sterile mortar, the roots were placed in sterile water and shaken to obtain an intraroot suspension. Finally, the intraroot suspension was diluted and plated. The growth of strain Z3-4 on the plate indicated that it could colonize the tobacco roots.

[0029] The present invention also provides a method for detecting the effect of the aforementioned *Pseudomonas mandelii* Z3-4 or the aforementioned bacterial agent or preparation on enhancing systemic immunity against bacterial wilt in tobacco:

[0030] (1) Preparation of root separation device: Two 500ml plastic cups were used to make a dual-chamber plant separation culture system, which was filled with sterile soil.

[0031] (2) Planting: Sterile tobacco seedlings cultured in MS medium for 14 days were planted in the central partition area. At the same time, the roots were evenly divided into two parts and planted in the two compartments. The seedlings were cultured for another 7 days to induce the roots to differentiate evenly in the two compartments.

[0032] (3) Inoculation of Z3-4 suspension: Fermentation of strain Z3-4 was carried out at 30℃ and 170r / min for 2 days, and the bacterial concentration was adjusted to OD using sterile water. 600 =0.5. Then, 10 ml of the above fermentation broth was inoculated into one compartment of the plant septate culture system, which was called the induction side. The control treatment was inoculated with an equal volume of sterile water on the induction side.

[0033] (4) Inoculation of pathogens: Seven days after Z3-4 treatment, 10 ml of Ralstonia solanacearum (OD600=0.5) was inoculated into the contralateral root compartment (systemic side) to simulate unilateral infection of the pathogen.

[0034] (5) Systemic immune resistance record: At the onset of bacterial wilt, a whole-plant survey was conducted on the control treatment and the Z3-4 inoculation treatment group. The survey was conducted every 3 days to accurately record the number of diseased plants and the disease severity, and to calculate the incidence rate, disease index and relative control efficacy.

[0035] (6) Calculation of resistance effect: Control effect % = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0036] This invention provides a plate confrontation culture method, which determines the ability of a bacterial strain to directly inhibit Ralstonia solanacearum by measuring the diameter of the inhibition zone.

[0037] (1) Culture and resuspension of strains: Z3-4 and bacterial wilt pathogens were inoculated into TSB liquid medium, fermented, centrifuged and resuspended.

[0038] (2) Pathogen coating: Add the bacterial suspension to the center of the TSA plate and spread it evenly on the entire plate surface with a sterile coating stick until the bacterial suspension is completely absorbed.

[0039] (3) Spot inoculation of strain Z3-4: Punch holes in the filter paper strip to form 6mm diameter discs, sterilize them, and place them in the middle of the TSA medium. Use a pipette to draw up the bacterial suspension of strain Z3-4 and drop it onto the filter paper strip.

[0040] (4) Inhibition zone determination: After 1-3 days of culture, the diameter of the inhibition zone (unit: mm) was determined by the cross-cross method. Each treatment was repeated 3 times. The larger the diameter, the stronger the antagonistic ability.

[0041] This invention also provides an application of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned inoculum or preparation in pot experiments to improve the control of tobacco bacterial wilt:

[0042] (1) Preparation of microbial suspension: Activate strain Z3-4, select a single colony, ferment strain Z3-4, centrifuge, discard the supernatant, resuspend the bacterial suspension with sterile water, and adjust OD600 to 1.0 to obtain the bacterial suspension required for inoculation.

[0043] (2) Potted planting: Uniformly growing Yunyan 87 seedlings were transplanted into pots containing an equal amount of sterilized potting soil. Each seedling was inoculated with a bacterial suspension at the root. The control group seedlings were inoculated with an equal amount of sterile water at the root. The bacterial suspension was inoculated again after transplanting. After transplanting, the pathogen solution was applied by root irrigation, and applied multiple times.

[0044] (3) Disease incidence statistics: Placed in a greenhouse with alternating light and dark conditions, the disease incidence was observed regularly, the disease index was counted, and the prevention and control effect was calculated.

[0045] The present invention also provides a field application of the aforementioned Pseudomonas mandelii Z3-4 or the aforementioned inoculum or preparation for the control of tobacco bacterial wilt.

[0046] (1) Preparation of microbial suspension: Z3-4 strain was activated, a single colony was selected, and Z3-4 strain was fermented for 2 days. After centrifugation, the supernatant was discarded, the bacterial suspension was resuspended with sterile water, and the OD600 was adjusted to 1.0 to obtain the bacterial suspension required for inoculation.

[0047] (2) Preparation of microbial agent: Weigh rice husk charcoal and plant organic fertilizer and mix them in a 1:1 ratio. Then, sterilize at high temperature and cool. Adjust the carrier moisture to a moist but non-clumping state and pack it into a self-sealing bag. Add Z3-4 bacterial suspension and mix thoroughly. Randomly punch holes in the bag as ventilation holes for microbial fertilizer fermentation. Then, put another self-sealing bag over it, punching multiple ventilation holes in different places. Place it in an incubator for cultivation and then remove it. Store at room temperature under shaded conditions.

[0048] (3) Field application: In 2024, two tobacco fields in Enshi City that had previously shown symptoms of bacterial wilt due to continuous cropping were selected as field test plots, with 20 mu (approximately 3.3 hectares) of demonstration area applied at each site. When transplanting tobacco seedlings, the above-mentioned inoculant was mixed with the local unsterilized tobacco topsoil and then evenly applied to the planting holes. The control group received no treatment, but all other field management practices were the same.

[0049] (4) Field survey: In early August 2024, the incidence rates of the treated plots and control plots were statistically analyzed to obtain the application effect of Z3-4 in the field.

[0050] Beneficial effects: Compared with existing technologies, this invention has the following significant advantages: A specific bacterial strain Z3-4 was isolated and screened from the roots of disease-resistant tobacco, which showed potential for biocontrol applications in pot experiments. Z3-4 efficiently colonizes and proliferates in tobacco roots and exhibits the ability to induce systemic disease resistance in tobacco plants, significantly improving the ability to control tobacco diseases. Attached Figure Description

[0051] Figure 1 The growth of Pseudomonas Z3-4 strain on TSA medium;

[0052] Figure 2 The colonization effect of Z3-4 and representative strains on sterile tobacco seedlings 4 weeks after inoculation;

[0053] Figure 3 Images used to verify the systemic control of bacterial wilt in tobacco plants by Pseudomonas Z3-4 strain through root division experiments;

[0054] Figure 4 Images used in plate antagonism assays to verify the direct inhibition of bacterial wilt by biocontrol strains on tobacco plants;

[0055] Figure 5 Images used in pot experiments to verify the control of bacterial wilt of tobacco plants by Pseudomonas Z3-4 strain. Detailed Implementation

[0056] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0057] Example 1:

[0058] (1) Aseptic root acquisition: Intact root systems of disease-resistant tobacco plants were collected from Hefeng County, Enshi City, Hubei Province. The collected tobacco roots were then placed in conical flasks filled with sterile water. The flasks were then subjected to ultrasonic vibration for three low-frequency ultrasonic cleanings to remove surface soil particles and most attached microorganisms. The material was then immersed in 75% ethanol for 3.0 min, followed by soaking in sodium hypochlorite solution with 3.5% active chlorine for 3.5 min, and finally washed five times in sterile water. After absorbing residual moisture with sterile absorbent paper, the roots were ground into powder using a sterile mortar.

[0059] (2) Coating separation: Weigh 0.1g of root powder into a 2ml sterile centrifuge tube, add 0.9mL of sterile water, and dilute twice to obtain a dilution of 10. -3 The soil dilution was spread at 0.1 mL onto TSA agar plates (agar composition: 5 g soybean peptone, 15 g tryptone, 5 g NaCl, 15 g agar, 1000 mL distilled water) and incubated at 26°C for 1-3 days.

[0060] (3) Cultivation and purification: Single colonies were picked and streaked onto isolation medium for purification culture. Three rounds of purification were performed in total. Figure 1 The strain obtained after purification and culture was used as the test strain and temporarily stored on TSA plates.

[0061]

[0062] (4) This strain belongs to the phylum Proteobacteria, class Gamma-Proteobacteria, order Pseudomonas, family Pseudomonasceae, genus Pseudomonas. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, on August 18, 2025, with accession number CGMCC No. 35660, and its taxonomic name is Pseudomonas mandelii.

[0063] Example 2: Colonization ability of Z3-4 on tobacco roots

[0064] Tobacco seeds were surface-sterilized with 75% ethanol (30 s) and 3% NaClO (15 min, 3 times), respectively, and then placed in half-strength MS medium (containing 2% (wt / vol) sucrose and 0.8% (wt / vol) agar). After 7 days of germination, seedlings were transferred to half-strength MS medium (preparation of half-strength MS medium: dissolve 2.2 g of Murashige Skoog medium powder (Haibo Biotechnology, catalog number HB8469-6) with 10 g of sucrose and 8 g of agar powder in 1 L of sterile deionized water, adjust the pH to 5.8, autoclave at 121℃ for 15 minutes, and dispense 50 ml into 13 cm Petri dishes in a laminar flow hood), with 2 seedlings inoculated into each medium. The plants were incubated at 22℃ for 16 h in light and 8 h in darkness for 7 days. Z3-4 biocontrol strain and two other representative strains were selected and fermented at 30℃ and 170 r / min. The bacterial concentration was adjusted to OD using sterile water. 600 =0.1. 50 μl of bacterial suspension was inoculated into each tobacco plant. Bacteria were recovered weekly for 3 weeks, 14–28 days post-inoculation. The tobacco roots were rinsed with sterile water to remove floating bacteria. The roots were then placed in PBS buffer for 5 min to extract bacteria from the rhizosphere and within the roots. After removing the roots, a rhizosphere suspension was obtained; the remaining roots were soaked in 75% ethanol for 5 min, followed by soaking in 3% NaClO solution for 3 min, and finally rinsed 5 times with sterile water. The roots were then ground in a sterile mortar and mixed with sterile water to obtain an intraroot suspension. The rhizosphere and intraroot suspensions were diluted and plated separately. The bacterial colonization in the roots was determined using Table 1 and... Figure 2 It can be seen that Z3-4 significantly proliferated in the rhizosphere and roots two weeks after inoculation. The colonization rate stabilized three weeks after inoculation, with the number of bacteria in the rhizosphere and roots increasing by approximately 5 and 10 times, respectively. The growth of strain Z3-4 on sterile seedlings indicates its ability to colonize the tobacco rhizosphere and roots. Furthermore, the colonization capacity of strain Z3-4, both in the rhizosphere and roots, was significantly higher than that of the other two representative biocontrol strains of Pseudomonas.

[0065] Table 1 shows the colonization rate of Z3-4 and representative strains on the tobacco root surface.

[0066]

[0067]

[0068] Example 3: Root division experiment to determine immune capacity

[0069] A dual-compartment plant culture system was constructed using two 500ml plastic cups filled with sterile soil. Sterile tobacco seedlings cultured in MS medium for 14 days were transplanted into the central compartment, while the root systems were evenly divided into two parts and planted in the two compartments. Cultured for another 7 days to induce uniform root differentiation in both compartments. Strains Z3-4 were fermented at 30℃ and 170 rpm for 2 days, and the bacterial concentration was adjusted to OD using sterile water. 600 =0.5. Then, 10 ml of the above fermentation broth was inoculated into one side compartment of the plant diaphragmatic culture system, referred to as the induction side. An equal volume of sterile water was inoculated into the opposite root compartment. Seven days after treatment Z3-4, the opposite root compartment (system side) was inoculated with Ralstonia solanacearum swu-101 (OD100). 600 =0.5, Regulation of the physiology and virulence of Ralstonia solanacearum by the second messenger 2′,3′-cyclic guanosine monophosphate. Nature Communications volume14, Article number:7654(2023) (Cite this article) 10ml, to simulate unilateral infection of pathogens.

[0070] At the onset of bacterial wilt, whole-plant surveys were conducted on the control group and the Z3-4 inoculation group every 3 days to accurately record the number of diseased plants and the disease severity, and to calculate the incidence rate, disease index and relative control efficacy.

[0071] Among them, the disease index = ∑(level number × number of diseased plants at that level) / (total number of plants surveyed × highest level number).

[0072] The results are as follows Figure 3 The results showed that the Z3-4 beneficial bacteria could enhance tobacco disease resistance by inducing systemic immunity. Compared with the control group, 30 days after inoculation with pathogens, the Z3-4 beneficial bacteria significantly slowed down and weakened plant disease symptoms, effectively inhibiting disease occurrence.

[0073] Example 4: Effects on the growth of pathogens

[0074] 1. Culture and resuspension of bacterial strains: Z3-4 and Ralstonia solanacearum swu-101 were inoculated into TSB liquid medium (medium composition: 5g soybean peptone, 15g tryptone, 5g NaCl, 1000mL distilled water) and fermented at 30℃ and 170r / min for 2 days. After centrifugation, the cultures were resuspended.

[0075] 2. Pathogen coating: Take 100 μL of bacterial wilt pathogen suspension (OD) 600 =0.6) Add to the center of the TSA plate and spread evenly over the entire plate surface with a sterile spreader until the bacterial solution is completely absorbed.

[0076] 3. Inoculation of strain Z3-4: Punch holes in filter paper strips to form 6mm diameter discs, sterilize them, and place them in the center of TSA medium. Use a pipette to draw 10ul (OD) of strain Z3-4 bacterial culture. 600 =1), drip onto the filter paper strip.

[0077] 4. Inhibition zone determination: Incubate at 26℃ for 1-3 days, and determine the diameter of the inhibition zone (unit: mm) using the cross-cross method. Each treatment is repeated 3 times. The larger the diameter, the stronger the antagonistic ability.

[0078] No direct inhibitory effect of Z3-4 on the pathogen was observed using the plate confrontation culture method; that is, no obvious inhibition zone was observed. Figure 4 This indicates that Z3-4 itself cannot inhibit bacteria, but it can activate plant immune resistance.

[0079] Example 5: Method for preparing microbial agents

[0080] First, activate strain Z3-4, select a single colony, and ferment strain Z3-4 at 30℃ and 170 r / min for 2 days. Centrifuge the bacterial solution at 6000 rpm for 5 min, discard the supernatant, resuspend the bacterial cells in sterile water, and then measure the OD value. 600 Adjust to 1.0 to obtain the desired bacterial suspension.

[0081] Preparation of microbial agent: Weigh rice husk charcoal (Ningbo Huacai Horticulture Co., Ltd., item number: D575864245120) and rapeseed cake fertilizer (Ningbo Huacai Horticulture Co., Ltd., item number: 002) and mix them in a 1:1 ratio by weight. Then, sterilize at 121℃ for 20 minutes, cool, and adjust the carrier moisture content to a moist but non-clumping state. Pack the mixture into a self-sealing bag. Add Z3-4 bacterial suspension at a ratio of 10% (mL / g) and mix thoroughly. Make holes in the bag for ventilation during fermentation. Then, place the bag inside another self-sealing bag, making multiple ventilation holes in different locations. Incubate at 28℃ for 3 days, then remove. Store at room temperature under shaded conditions.

[0082] Example 6: Pot experiment demonstrating the effect of biocontrol strains on the control of bacterial wilt in tobacco plants

[0083] (1) Preparation of tobacco seedlings: After high-temperature and high-pressure sterilization of the substrate, it is placed in a sterile floating plate and leveled. Two seeds are sown in each hole, and the seedling trays are placed in a climate chamber for cultivation. The cultivation conditions are: light-dark treatment 12L:12D, temperature 28℃. After 1-2 weeks of cultivation, depending on the growth status of the tobacco plants (i.e., when growth is stable), seedlings are removed, leaving one tobacco plant in each hole. When removing seedlings, care should be taken to retain seedlings that are growing well and are of stable size.

[0084] (2) Obtaining sterile soil: The soil used for potted plants was collected from healthy tobacco fields in Hubei Province. After removing impurities, the soil was sieved. The tobacco field soil was placed in sterilization bags and sterilized under high temperature and high pressure for later use.

[0085] (3) Preparation of microbial suspension: Z3-4 strain was activated, single colonies were selected, and cultured in TSB medium at 160 rpm and 26℃ for 16-18 h with shaking. The bacterial suspension was then centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the bacterial suspension was resuspended in sterile water. The OD value was then adjusted. 600 Adjust to version 1.0 to obtain the bacterial solution required for inoculation.

[0086] (4) Potted Planting: Uniformly growing Yunyan 87 seedlings were transplanted into pots containing an equal amount of sterilized potting soil. Each seedling was inoculated with 10 mL of bacterial suspension at the base of its roots. Control group seedlings were inoculated with an equal amount of sterile water at the base of their roots. The seedlings were inoculated with the bacterial suspension twice more, at 5 and 10 days after transplanting. Twelve days after transplanting, the seedlings were inoculated with Ralstonia solanacearum (OD2000). 600 =0.5)10ml, applied once every 5 days, for a total of 3 applications of pathogenic bacterial solution. The experiment was set up with 4 replicates, each containing 10 seedlings. Tobacco was cultured in a sterile room, and the plants were placed randomly with their positions changed periodically.

[0087] (5) At the initial stage of bacterial wilt, conduct a whole-plant survey of each replicate every 3 days, accurately record the number of infected plants and the severity level, and calculate the incidence rate, disease index, and relative control efficacy. The disease severity grading standard is based on the method of Fang Shumin et al.: Grade 0: No disease in tobacco plants; Grade I: A few leaves at the base of the tobacco plant are infected, with slight wilting or occasional chlorotic streaks at the base; Grade II: Most leaves in the middle and lower parts of the tobacco plant are infected and wilted, or there are obvious black streaks at the base of the stem; Grade III: More than 2 / 3 of the leaves of the tobacco plant are infected and wilted, or the black streaks at the base of the stem extend to the top; Grade IV: The entire plant wilts or dies. The disease index, incidence rate, and control efficacy are calculated using the following formulas:

[0088] Disease index = ∑(level number × number of diseased plants at that level) / (total number of plants surveyed × highest level number)

[0089] Incidence rate % = (Number of infected plants / Number of plants surveyed) × 100%

[0090] Prevention and control efficacy % = (Control disease index - Treatment disease index) / Control disease index × 100%

[0091] The results are as follows Figure 5 As shown, compared with the control group, the beneficial bacteria Z3-4 significantly reduced the disease index and effectively inhibited the occurrence of diseases.

[0092] Example 7: Field Application Results

[0093] (1) Preparation of microbial suspension: The Z3-4 strain was activated, single colonies were selected, and the Z3-4 strain was fermented at 30℃ and 170r / min for 2 days. The bacterial suspension was centrifuged at 6000rpm for 5min, the supernatant was discarded, and the bacterial suspension was resuspended in sterile water. The OD was then measured. 600 Adjust to version 1.0 to obtain the bacterial solution required for inoculation.

[0094] (2) Preparation of microbial agent: Weigh rice husk charcoal and plant organic fertilizer and mix them in a 1:1 ratio. Then, sterilize the mixture at 121℃ for 20 minutes and cool it. Adjust the moisture content of the carrier until it is moist but not clumpy, and pack it into a self-sealing bag. Add Z3-4 bacterial suspension at a ratio of 10% and mix thoroughly. Make holes in the bag as ventilation holes for microbial fertilizer fermentation. Then, put another self-sealing bag on the outside and make multiple ventilation holes in different places. Place it in an incubator and incubate at 28℃ for 3 days. Then, remove it from the incubator. Store it at room temperature under dark conditions.

[0095] (3) Field application: In 2024, two tobacco fields in Enshi City that had previously shown symptoms of bacterial wilt due to continuous cropping were selected as field test plots, with 20 mu (approximately 1.3 hectares) of demonstration area per plot. When transplanting tobacco seedlings, the above-mentioned inoculant was mixed with local unsterilized tobacco topsoil at a ratio of 1:1 (V:V) and applied evenly to the planting holes, 30g per hole. The control group received no treatment, but all other field management practices were the same.

[0096] (4) Field survey: In early August 2024, the incidence rates of the treated plots and control plots were statistically analyzed to obtain the application effect of Z3-4 in the field.

[0097] Table 2Z3-4 Effects of microbial inoculant application on economic traits of tobacco

[0098]

[0099] Table 2 shows that after the application of Z3-4 inoculant, compared with the control group, the incidence and incidence index of bacterial wilt were significantly reduced, the yield reduction in tobacco fields caused by bacterial wilt was significantly improved, and economic losses were reduced.

Claims

1. A strain of Pseudomonas moniliforme ( Pseudomonas mandelii Z3-4, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 35660, deposited on August 18, 2025, and classified as follows: Pseudomonas mandelii .

2. A microbial agent or preparation, characterized in that, The bacterial agent or preparation contains *Pseudomonas moniliforme* as described in claim 1. Pseudomonas mandelii Z3-4 or its fermentation broth or bacterial suspension, or one or more thereof.

3. The *Pseudomonas moniliforme* as described in claim 1 ( Pseudomonas mandelii The application of the fungal agent or preparation described in claim 2 or Z3-4 in the prevention and control of tobacco bacterial wilt disease.

4. The application according to claim 3, characterized in that, Methods for controlling tobacco bacterial wilt include applying the *Pseudomonas moniliforme* strain described in claim 1 to tobacco plants. Pseudomonas mandelii Z3-4 or the bacterial agent or preparation described in claim 2.

5. The application according to claim 3, characterized in that, The aforementioned bacterial wilt disease in tobacco is caused by Ralstonia solanacearum or its variants.

6. The application according to claim 3, characterized in that, Methods to prevent and control tobacco bacterial wilt include soaking tobacco seeds or drenching the roots of tobacco plants.

7. The *Pseudomonas moniliforme* as described in claim 1 (… Pseudomonas mandelii The use of the fungal agent or preparation described in Z3-4 or claim 2 in the preparation of products for the prevention and control of tobacco bacterial wilt.

8. The application according to claim 7, characterized in that, The products include vaccines.

9. The *Pseudomonas moniliforme* as described in claim 1 ( Pseudomonas mandelii The application of the microbial agent or preparation described in claim 2 or Z3-4 in inducing defensive metabolism in tobacco roots.

10. The *Pseudomonas moniliforme* as described in claim 1 (… Pseudomonas mandelii The application of the fungal agent or preparation described in claim 2 or Z3-4 in improving the immunity of tobacco to tobacco bacterial wilt disease.

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